msc enumeration kit human

For 25 tests Order no. 130-106-646

MSC Enumeration Kithuman

Miltenyi Biotec B.V. & Co. KG Friedrich-Ebert-Straße 68, 51429 Bergisch Gladbach, GermanyPhone +49 2204 8306-0, Fax +49 2204 85197

[email protected], www.miltenyibiotec.com

140

-00

4-7

78

.02

Unless otherwise specifi cally indicated,

Miltenyi Biotec products and services are for research use only

and not for diagnostic or therapeutic use.

1. DescriptionContents

2 3140-004-778.02 140-004-778.02

Contents

1. Description

1.1 Principle of the assay

1.2 Background information

1.3 Application

1.4 Description of kit components

1.5 Reagents and instruments requirements

2. Protocols

2.1 Immunofl uorescent labeling

2.2 Flow cytometric acquisition and data analysis using

the MACSQuant® Analyzer 10

2.3 Flow cytometric acquisition and data analysis using

fl ow cytometer other than MACSQuant® Analyzer 10

3. Appendix

4. References

1. Description

Components 250 μL MSC Staining Cocktail, human:

cocktail of fl uorochrome-conjugated monoclonal

antibodies: CD45-FITC (clone: 5B1, isotype: mouse

IgG2a), CD235a (Glycophorin A)-VioBlue® (clone:

REA175, isotype: recombinant human IgG1),

CD271 (LNGFR)-PE (clone: Me20.4-1H.4, isotype:

mouse IgG1).

250 μL Anti-MSCA-1 (W8B2)-APC, human:

anti-MSCA-1 (W8B2) monoclonal antibody (clone:

W8B2, isotype: mouse IgG1) conjugated to APC.

250 μL MSC Isotype Cocktail, human:

cocktail of Mouse IgG1-APC (clone: IS5-21F5.12.2)

and f luorochrome-conjugated monoclonal

antibodies: CD45-FITC (clone: 5B1, isotype: mouse

IgG2a), CD235a (Glycophorin A)-VioBlue (clone:

REA175, isotype: recombinant human IgG1),

CD271 (LNGFR)-PE (clone: Me20.4-1H.4, isotype:

mouse IgG1).

5 mL Red Blood Cell Lysis Solution (10×)

1 mL FcR Blocking Reagent, human

500 μL 7-AAD Solution (52.5 μg/mL)

Capacity For 25 tests with 200 μL human cell sample per test.

Product format MSC Staining Cocktail, Anti-MSCA-1 (W8B2)-

APC, MSC Isotype Cocktail, and FcR Blocking

1. Description1. Description

4 5140-004-778.02 140-004-778.02

Reagent are supplied in buff er containing stabilizer

and 0.05% sodium azide.

Storage Store all components protected from light at 2–8 °C.

Do not freeze. Th e expiration date is indicated on

the vial label.

1.1 Principle of the assay

The MSC Enumeration Kit has been designed for the quantification of

mesenchymal stromal cells (MSCs) in human bone marrow samples,

based on the expression of CD271 (LNGFR) and Anti-MSCA-1 (W8B2).

The kit allows the identification of CD45+ leukocytes and CD271

(LNGFR)+/Anti-MSCA-1 (W8B2)+ MSCs, which are also CD45dim.

CD235a (Glycophorin A) is used to exclude remaining red blood cells,

which were not completely lysed by the Red Blood Cell Lysing Solution.

For one test, two 100 μL samples of bone marrow are needed.

The MSCs are detected by staining one 100 μL sample with the

MSC Staining Cocktail as well as with Anti-MSCA-1 (W8B2)-APC.

Subsequently, erythrocytes are lysed using the Red Blood Cell Lysis

Solution.

Additionally, a 100 μL control sample is stained with the MSC Isotype

Cocktail. This is required for the proper setting of relevant gates during

flow cytometry analysis.

The FcR Blocking Reagent increases the specificity of immunofluorescent

staining.

Dead cells are excluded from the analysis by adding of DNA stain

7-aminoactinomycin D (7-AAD). 7-AAD diffuses through the cell

membrane of dead cells and intercalates with their DNA.

Finally, cells are analyzed with flow cytometers able to detect 4-color

fluorescence (488 nm argon laser, 635 nm red diode laser, optional:

405 nm violet laser). The use of the MACSQuant® Flow Cytometers

enable an automated and accurate cell count. If using flow cytometers

other than the MACSQuant Flow Cytometer, it is necessary to determine

the absolute cell count by analysis of commercially available counting

particles such as present in BD Trucount™ Tubes.

1.2 Background information

The large scale isolation and expansion of MSCs from human bone

marrow is time consuming, cost effective, and highly dependent on the

quality of the starting material. Therefore it is desirable to qualify the

bone marrow source with regard to MSC content. The common way

to quantify MSCs within human blood sources is the colony forming

unit-fibroblast (CFU-F) assay, which is time consuming, dependent on

serum lots, and plating densities, as well as considerably subjective in

defining and scoring colonies. It was shown that Anti-MSCA-1 (W8B2)

only detects CD271 (LNGFR)bright cells and only these cells give rise to

CFU-F1. A close linear relationship was observed between the number

of CFU-F colonies counted manually after 14 days of culture and the

number of CD271 (LNGFR)bright cells per milliliter of aspirate2.

1.3 Application

● Enumeration of MSCs in human bone marrow samples

1. Description1. Description

6 7140-004-778.02 140-004-778.02

Red Blood Cell Lysis Solution

Upon incubation of the cells in 1× Red Blood Cell Lysis Solution

erythrocytes are selectively lysed. Dilute Red Blood Cell Lysis Solution

(10×) 1:10 with double-distilled water (ddH2O).

1.5 Reagents and instruments requirements

● Double-distilled water (ddH2O)

● 5 mL tubes

● Micropipettes with tips

● Centrifuge (2–8 °C)

● Vortex mixer

● Rotation device for tubes: MACSmix™ Tube Rotator (# 130-090-753)

● Th ree-laser fl ow cytometer and soft ware (405 nm, 488 nm, and

638 nm laser), e.g., MACSQuant Analyzer 10 (# 130-096-343) with

MACSQuantify™ Soft ware.

▲ Note: Alternatively, a dual-laser fl ow cytometer and soft ware (488 nm and 638 nm

laser) can be used. Exclusion of remaining red blood cells using CD235a is then not

possible.

● (Optional) BD Trucount Absolute Counting Tubes (# 340334)

● (Optional) MACS® SmartStrainers (100 μm) (# 130-098-463)

● (Optional) MACS Comp Bead Kit, anti-mouse Igκ (# 130-097-900)

● (Optional) MACS Comp Bead Kit, anti-REA (# 130-104-693)

1.4 Description of kit components

MSC Staining Cocktail

Components of the MSC Staining Cocktail, human

Antibody Fluorochrome Specifi city

CD45 FITC Leukocytes

CD235a (Glycophorin A) VioBlue Erythrocytes

CD271 (LNGFR) PE MSCs, lymphocyte progenitors

Fluorescein (FITC), Phycoerythrin (PE)

Anti-MSCA-1 (W8B2)-APC


Anti-MSCA-1 (W8B2) APC MSCs

Allophycocyanin (APC)

MSC Isotype Cocktail

Components of the MSC Isotype Cocktail, human


CD45 FITC Leukocytes

CD235a (Glycophorin A) VioBlue Erythrocytes

CD271 (LNGFR) PE MSCs, lymphocyte progenitors

Mouse IgG1 APC Isotype control

2. Protocol2. Protocol

8 9140-004-778.02 140-004-778.02

2. Protocols

Th e following protocol has been optimized for mesenchymal stromal

cell (MSC) enumeration in a human bone marrow sample. For best

results use starting material directly aft er aspiration.

▲ Bone marrow samples have to be supplemented with an anticoagulant,

for example, heparin.

▲ Bone marrow samples should be stored at room temperature under

agitation until analysis.

▲ When using bone marrow aspirate pass the material through a

MACS SmartStrainer (100 μm).

▲ Dilute Red Blood Cell Lysis Solution (10×) 1:10 with double-distilled

water (ddH2O), for example, dilute 200 μL of Red Blood Cell Lysis

Solution (10×) with 1800 μL of ddH2O. Do not use deionized water!

Store the prepared 1× Red Blood Cell Lysis Solution at room temperature.

Discard unused solution at the end of the day.

2.1 Immunofl uorescent labeling

▲ Volumes given below are for one test with two 100 μL samples ofbone marrow.

1. Label two tubes with A (control sample) and B (MSC sample).

▲ Note: When using other fl ow cytometer than the MACSQuant Flow Cytometers

use two BD Trucount Absolute Counting Tubes.

2. Carefully pipette 100 μL of well-mixed human bone marrow to the

bottom of each tube.

▲ Note: For each sample tube use a fresh micropipette tip.

3. Add 20 μL FcR Blocking Solution into each tube.

4. Add 10 μL of MSC Isotype Cocktail into tube A and 10 μL of MSC

Staining Cocktail as well as 10 μL of Anti-MSCA-1 (W8B2)-APC

into tube B.

5. Immediately vortex thoroughly and incubate for 10 minutes at

2–8 °C in the dark.

6. Add 860 μL of 1× Red Blood Cell Lysis Solution into tube A and

850 μL in tube B.

7. Immediately vortex and incubate for 10 minutes at room

temperature in the dark using the MACSmix Tube Rotator.

8. Add 10 μL of the 7-AAD Solution into each tube.

9. Directly proceed with fl ow cytometric analysis.

▲ Note: Analysis should be performed within 20 minutes aft er staining.


10 11140-004-778.02 140-004-778.02

2.2 Flow cytometric acquisition and data analysis using the

MACSQuant® Analyzer 10

Before starting to acquire the samples make sure that the MACSQuant®

Analyzer 10 fl ow cytometer has been carefully cleaned in order to allow

the detection of rare MSCs.

Instrument setting and data acquisition

Choose an instrument setting for standard four-color cell analysis of

human blood leukocytes. A proper instrument setting can be reached

by adjusting parameters with separate FITC-, PE-, VioBlue-, and

APC-conjugated antibodies or by using the MACS Comp Bead Kit,

anti-mouse Igκ or the MACS Comp Bead Kit, anti-REA.

▲ Exclude debris from data acquisition by an appropriate value for the

threshold on forward scatter (FSC).

Creation of dot plots

For data acquisition and analysis create the following dot plots (region

settings are shown in the examples below):

A Plot: Forward scatter (FSC) versus side scatter (SSC)

B Plot: 7-AAD Solution versus SSC

C Plot: FSC versus CD235a (Glycophorin A)-VioBlue

D Plot: CD45-FITC versus CD271 (LNGFR)-PE

E Plot: Mouse IgG1-APC versus CD271 (LNGFR)-PE and Anti-MSCA-1

(W8B2)-APC versus CD271 (LNGFR)-PE

Defi ne the experiment as follows:

Experiment setup

Rack type Chill 5 Tube Rack, choose 2 positions

Sample ID 1. position: Control sample

2. position: MSC sample

Flow rate medium

Mode standard

Uptake volume 450 μL

Sample volume 1 mL

Instrument setting PBMC compensation (needs to be performed before data anaylsis)*

Annotations 1. position: CD45-FITC, CD235a (Glycophorin A)-VioBlue, CD271 (LNGFR)-PE, Mouse IgG1-APC, 7-AAD Solution

2. position: CD45-FITC, CD235a (Glycophorin A)-VioBlue, CD271 (LNGFR)-PE, Anti-MSCA-1 (W8B2)-APC, 7-AAD Solution

* Please refer to the user guide of the MACSQuantify Soft ware for detailed instructions.

2.2.1 Flow cytometric data acquisition


12 13140-004-778.02 140-004-778.02

B Plot: 7-AAD Solution versus SSC (gated on P1)

Draw region P2 to exclude dead cells.

10³-10

10¹ 10²0

25

50

75

100

1

7-AAD

Sid

e s

catt

er

P2

C Plot: FSC versus CD235a (Glycophorin A)-VioBlue (gated on P1/P2)

Draw region P3 to exclude CD235a+ erythrocytes.

10³

-10

10¹

10²

0

250 500 750 1000

1

Forward scatter

CD

23

5a

-Vio

Blu

e

P3

▲ Th e gating strategy below describes the fl ow cytometric analysis of a

bone marrow sample using the MACSQuantify Soft ware.

▲ Perform the analysis of the MSC sample stained with the MSC

Staining Cocktail in the same way as the analysis of the control sample

stained with the MSC Control Cocktail.

MSC sample

A Plot: FSC versus SSC (no gate)

Draw region P1 to exclude debris and platelets.

Forward scatter

Sid

e s

catt

er

P1

2.2.2 Flow cytometric analysis


14 15140-004-778.02 140-004-778.02

D Plot: CD45-FITC versus CD271 (LNGFR)-PE (gated on P1/P2/P3)

Defi ne region P4 thereby including all CD271 (LNGFR)+ cells.

10³-101

10¹ 10²0

10³

10²

10¹

CD45-FITC

CD

27

1 (

LN

GF

R)-

PE

-1 1

P4

E Plot: Mouse IgG1-APC/Anti-MSCA-1 (W8B2)-APC versus CD271

(LNGFR)-PE (gated on P1/P2/P3/P4)

Copy all gates into the control sample.

Defi ne region P5 with the control sample to include all Anti-MSCA-1

(W8B2)-APC+ events within the MSC sample. It is important to set gate

P5 as close as possible to the Anti-MSCA-1 (W8B2)– events to include all

Anti-MSCA-1 (W8B2)+ and Anti-MSCA-1 (W8B2)dim+ cells.

Control sample MSC sample

10³-101

10¹ 10²0

10³

10²

10¹

Mouse IgG1-APC/Anti-MSCA-1-APC

CD

27

1 (

LN

GF

R)-

PE

-1 1

P50.00%-2

1

2.22/mL

10³-101

10¹ 10²0

10³

10²

10¹


CD

27

1 (

LN

GF

R)-

PE

-1 1

P50.02%-2

175

389/mL


16 17140-004-778.02 140-004-778.02

For the calculation the gate statistic of plot E of the MSC sample has

been used. Following region functions are needed:

Parameter Description Example

a Count/mL Number of events per mL staining volume 389/mL

b %–2 Percent grand parent, for information only 0.02%

c Staining volume

Complete volume after labeling including cocktails, antibody, Red Blood Cell Lysis Solution, FcR Blocking Reagent, and 7-AAD Solution (1000 μL in standard applications)

1000 μL

d Sample volume

Bone marrow volume used for one staining (100 μL in standard applications)

100 μL

Calculation of dilution factor

Parameter Calculation Example

e Dilution factor c / d 1000 μL/100 μL = 10

Calculation of MSC number


f Number of MSCs/mL bone marrow

a × e 389/mL × 10 = 3890/mL

In the given example, the number of MSCs is 3890/mL human bone

marrow aspirate.

2.3 Flow cytometric acquisition and data analysis using fl ow

cytometer other than MACSQuant® Analyzer 10

Before starting to acquire the samples make sure that the fl ow cytometer

has been carefully cleaned in order to allow the detection of rare MSCs.

Instrument setting and data acquisition

Choose a fl ow cytometer instrument setting for standard four-color cell

analysis of human blood leukocytes. A proper instrument setting can

be reached by adjusting parameters with separate FITC-, PE-, and APC-

conjugated antibodies.

▲ Exclude debris from data acquisition by an appropriate value for the

threshold on forward scatter (FSC).

▲ A minimum of 100 CD271 (LNGFR)+ Anti-MSCA-1 (W8B2)+ events

should be counted. Additionally a minimum of 1000 counting particle

or a maximum of 10 minutes acquisition time should be analyzed.

Creation of dot plots

For data acquisition and analysis create the following dot plots (region

settings are shown in the examples below):

A Plot: Forward scatter (FSC) versus side scatter (SSC)

B Plot: 7-AAD Solution versus SSC

2.3.1 Flow cytometric data acquisition

2.2.3 Determination of MSC number


18 19140-004-778.02 140-004-778.02

C Plot: CD45-FITC versus CD271 (LNGFR)-PE

D Plot: Mouse IgG1-APC versus CD271 (LNGFR)-PE and Anti-MSCA-1

(W8B2)-APC versus CD271 (LNGFR)-PE

E Plot: CD45-FITC versus CD271 (LNGFR)-PE

F Plot: Time versus CD271 (LNGFR)-PE

Defi nition of logical gates

Defi ne and label the gates described in the following table:

Gate Label Defi nition

G1 WBCs R1

G2 Viable WBCs R1*R2

G3 CD271 (LNGFR)+ R1*R2*R3

G4 CD271 (LNGFR)+ aMSCA-1+ R1*R2*R3*R4

G5 R5

G6 Counting beads R5*R6

▲ Th e general gating strategy below describes the fl ow cytometric

analysis of a bone marrow sample when using another fl ow cytometer

than the MACSQuant® Analyzer 10.

▲ Perform the analysis of the MSC sample stained with the MSC

Staining Cocktail in the same way as the analysis of the control sample

stained with the MSC Control Cocktail.

MSC sample

A Plot: FSC versus SSC (no gate)

Draw region R1 to exclude debris and platelets.

Forward scatter

Sid

e s

catt

er

R1

2.3.2 Flow cytometric analysis


20 21140-004-778.02 140-004-778.02

B Plot: 7-AAD Solution versus SSC (gated on R1)

Draw region R2 to exclude dead cells.

7-AAD

Sid

e s

catt

er

R2

C Plot: CD45-FITC versus CD271 (LNGFR)-PE (gated on R1/R2)

Draw region R3 thereby including all CD271 (LNGFR)+ cells. Generate a

gate statistic for later calculation.

CD45-FITC

CD

27

1 (

LN

GF

R)-

PE

R3

D Plot: Mouse IgG1-APC/Anti-MSCA-1 (W8B2)-APC versus CD271

(LNGFR)-PE (gated on R1/R2/R3)

Copy all gates into the control sample fi le.

Defi ne region R4 with the control sample to include all Anti-MSCA-1

(W8B2)-APC+ events within the MSC sample. It is important to set gate

R4 as close as possible to the Anti-MSCA-1 (W8B2)– events to include all

Anti-MSCA-1 (W8B2)+ and Anti-MSCA-1 (W8B2)dim+ cells.

Control sample MSC sample


CD

27

1 (

LN

GF

R)-

PE

R4


22 23140-004-778.02 140-004-778.02

E Plot: CD45-FITC versus CD271 (LNGFR)-PE (no gate)

Defi ne region R5 by including all FITC and PEbright counting beads.

R5

CD45-FITC

CD

27

1 (

LN

GF

R)-

PE

F Plot: Time versus CD271 (LNGFR)-PE (gated on R5)

Defi ne region R6 by including all monodisperses and bidisperses–

counting beads. Generate a gate statistic for later calculation.

Time

CD

27

1 (

LN

GF

R)-

PE

R6

For the calculations the gate statistics of plot C and F are used. A

summary of these statistics are shown in the table below.

Gate Events Gated [%]

G1 (WBCs) 140141 100.00

G2 (Viable WBCs) 140141 100.00

G3 (CD271 (LNGFR)+) 7731 5.52

G4 (CD271 (LNGFR)+ aMSCA-1+) 114 0.08

G5 16530 100.00

G6 (Counting beads) 16040 97.04

2.3.3 Analysis of the results using counting particles

3. Appendix2. Protocol

24 25140-004-778.02 140-004-778.02

Calculation of dilution factor

Th e dilution factor is calculated by dividing the staining volume (c,

complete volume aft er labeling) by the sample volume (d, bone marrow

volume used for one staining). Refer to section 2.2.3 for details.


Dilution factor c / d 1000 μL / 100 μL = 10

Calculation of MSCs number

Parameter Calculation

Number of MSCs/mL bone marrow

G4 × total counting beads per tube*

G6× dilution factor

Example:

114 × 50950

16040×10 = 3621/mL

* As indicated in the manufacturer`s instruction.

In the given example, the number of MSCs is 3621/mL human bone

marrow aspirate.

3. Appendix

MSCA-1 (W8B2) is identical to the tissue non-specifi c alkaline

phosphatase (TNAP). It was demonstrated that only MSCA-1+ but not

MSCA-1– bone marrow cells express the enzymatically active form of

the TNAP. In addition, it was shown in diff erent bone marrow sources

that only MSCA-1+ cells gave rise to MSCs3 as defi ned by the ISCT

consortium4. Nevertheless there are further MSC specifi c antibodies

which can be used in combination with CD271 (LNGFR) for MSC

detection and enumeration. Th e following table lists some of these

markers with suggested titers that can be used within this assay instead

of Anti-MSCA-1 (W8B2)-APC.

Specifi city Titer Synonym Order number

CD73-APC 1:40 ecto-5'-nucleotidase # 130-095-183

CD90-APC 1:40 Thy-1 # 130-095-402

CD140b-APC 1:10 PDGF-receptor # 130-105-280

4. References

26 27140-004-778.02 140-004-778.02

4. References

1. Bühring, H. J. (2007) Novel markers for the prospective isolation of human MSC.

Ann. N. Y. Acad. Sci. 6: 262–271.

2. Cuthbert, R. (2012) Single-platform quality control assay to quantify multipotential

stromal cells in bone marrow aspirates prior to bulk manufacture or direct

therapeutic use. Cytotherapy 14( 4):431–440.

3. Sobiesiak, M. et al. (2010) Th e mesenchymal stem cell antigen MSCA-1 is identical to

tissue non-specifi c alkaline phosphatase. Stem Cells Dev. 19(5): 669–677.

4. Dominici, M. (2006) Minimal criteria for defi ning multipotent mesenchymal stromal

cells. Th e International Society for Cellular Th erapy position statement. Cytotherapy

8 (4): 315–317.

Refer to www.miltenyibiotec.com for all data sheets and protocols.

Miltenyi Biotec provides technical support worldwide. Visit

www.miltenyibiotec.com for local Miltenyi Biotec Technical Support

contact information.

WarningsReagents contain sodium azide. Under acidic conditions sodium azide yields hydrazoic

acid, which is extremely toxic. Azide compounds should be diluted with running water

before discarding. Th ese precautions are recommended to avoid deposits in plumbing

where explosive conditions may develop.

28 29140-004-778.02 140-004-778.02

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and/or may have certain limitations. Certain uses may be excluded by separate terms

and conditions. Please contact your local Miltenyi Biotec representative or visit Miltenyi

Biotec’s website at www.miltenyibiotec.com for more information.

Th e purchase of this product conveys to the customer the non-transferable right to use

the purchased amount of the product in research conducted by the customer (whether

the customer is an academic or for-profi t entity). Th is product may not be further sold.

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30 31140-004-778.02 140-004-778.02

msc enumeration kit human

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